The molecular chaperone calnexin associates with the vacuolar H(+)-ATPase from oat seedlings.
Acidification of endomembrane compartments by the vacuolar-type H(+)-ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V1) and a membrane integral sector (V0); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein-protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V1V0-ATPases at the endoplasmic reticulum.
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Department of Plant Biology, University of Maryland, College Park 20742, USA.
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D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D029681:Arabidopsis Proteins; D018554:Avena; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D037281:Calnexin; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000737:chemistry; D008861:Microsomes / Q000737:chemistry; D008954:Models, Biological; D018832:Molecular Chaperones / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012995:Solubility; D025262:Vacuolar Proton-Translocating ATPases
D000911:Antibodies, Monoclonal; D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018832:Molecular Chaperones; D010940:Plant Proteins; C077221:BIP protein, Arabidopsis; D037281:Calnexin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases